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    题名: 酵母菌IDS2基因在大腸桿菌中的蛋白質表現和純化以及甲基化活性
    其它题名: Expression, purification, and methylation activity of yeast IDS2p in escherichia coli
    作者: 林榆清;Lin, Yu-chin
    贡献者: 淡江大學生命科學研究所碩士班
    陳銘凱;Chern, Ming-kai
    关键词: IDS2;啤酒酵母菌;甲基轉移酶;IEF;IDS2;ISaccharomyces cerevisiae;methyltransferase;IEF
    日期: 2009
    上传时间: 2010-01-11 02:30:47 (UTC+8)
    摘要: 蛋白質會由許多種方式來修飾,其中一種為蛋白質的甲基化,而形成蛋白質甲基化的酵素為甲基轉移酶,甲基轉移酶是將S-adenosylmethionine (S-腺甲硫氨酸)所提供的甲基轉移到蛋白質上。蛋白質的甲基化作用是一種轉譯後修飾常發生於polypeptide chain,它會調節生物的生理作用,包括DNA、RNA的代謝、蛋白質的生合成及訊息傳導。

    Ime2p是只在啤酒酵母菌(Saccharomyces cerevisiae)進行減數分裂時才會表現的protein kinase,而Ime2p會刺激早期、中期、晚期的減數分裂基因的表現,IDS2則是刺激Ime2p作用的基因,兩者之間在功能上相互影響。

    而根據以往文獻的報導,IDS2p具有甲基轉移酶之特徵序列。我們將啤酒酵母菌(Saccharomyces cerevisiae)IDS2基因建構到大腸桿菌的Rosseta 菌株中作蛋白質表現,利用His-tag融合純化出所需要的IDS2p,並及將△IDS2菌株之蛋白質抽取液作受質,加入氚化甲基S-腺苷甲硫氨酸[C3H3]S-adenosy-L-methionine一起進行甲基化反應,接著將受質蛋白質加以分離。將受質蛋白質以直立式凝膠電泳isoelectric focusing (IEF)進行第一維的分離,然後將分離的受質蛋白質取下進行SDS-PAGE的第二維電泳,最後我們將分離出的受質蛋白質取下進行質譜儀的鑑定分析。
    Proteins can be modified in several ways by the addition of methyl groups from S-adenosylmethionine. Methylation regulates a variety of biological functions including DNA and RNA metabolism, protein synthesis and signal transduction.

    Ime2p is a protein kinase that is expressed only during meiosis in Saccharomyces cerevisiae. Ime2p stimulates early, middle, and late meiotic gene expression. IDS2 (for IME2-dependent signaling) has a functional interaction with Ime2p.

    According to recent studies, the sequence IDS2, is homologous to a methyltransferase. In our studies, we constructed the sequence IDS2 of Saccharomyces cerevisiae and overexpressed IDS2p in E. coli Rosetta (DE3) strains, and purified IDS2p with His-tag. The protein extract from △IDS2 strain was methylated with the cosubstrate [C3H3]S-adenosy-L-methionine prior to separation. The labeled proteins were first separated by isoelectric focusing (IEF) on a vertical slab gel, and we cutted off the spot with labeled protein and separated the protein on SDS-PAGE. Finally the labeled protein was extracted from SDS-PAGE and identified by mass spectrometry.
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